Cultivation and Properties of Thiovulum niajus Hinze 63 



METHODS 



Enrichment Propagation of Crude Cultures 



Primary elective cultures were prepared by Wijler's method. 

 The trickle of seawater was initiated after about one to two weeks, 

 when the algae had started to decay and a thick film of bacterial 

 growth had developed on the surface; Thiovulum usually ap- 

 peared during the third week. Transfers were made to jars of 

 5-10 L capacity to which, depending on the condition of the cul- 

 ture, 5-20 ml of a saturated HjS solution were added once or 

 twice daily. For propagation on a smaller scale a 10 L resei*voir 

 with an aerated solution of 0.005% NH4CI, 0.005% KH.PO^ and 

 0.005% Na2C03 in seawater, pH 8, was attached to a continuous 

 culture device as described by Bulder (4), set at a delivery rate 

 of 20-35 ml/hr. The medium flowed through a 1 L bottle without 

 air space to wliich 2-5 ml of a saturated HlS solution were added 

 daily from a burette, permanently connected to an HlS generator. 

 It then entered at the bottom of a 100 ml Erlenmeyer flask pro- 

 vided with a side tube near the top; the overflow dripped into a 

 second similar flask, and from here into a sink. The flasks served 

 as culture vessels. The entire system was placed in a 15 C incu- 

 bator. 



Purification of Inoculum 



About 50-100 ml of a Thiovulum suspension, obtained from 

 veils in a primary or secondary culture, were rapidly filtered 

 tlirough a thin layer of cotton into a 100 ml glass cylinder. After 

 five to fifteen minutes the cells settled in a characteristic pattern 

 on or near the bottom. They were removed in as small a volume 

 as possible, and transferred to a test tube with 15 ml sterile sea- 

 water where, in another five to fifteen minutes, they formed a 

 new web near the bottom. Subsequent transfers, with sterile 

 pipettes, completed the operations. A healthy culture could be 

 so "washed" up to ten times. Visual observation of the settling 

 process was facilitated by lateral illumination with a point source. 

 Plating of 1 ml samples from successive tubes in 0.1 per cent 

 peptone seawater agar, incubated at 22 C, served as a check on 

 the efiiciency of the procedure in the elimination of heterotrophs. 



